Ishrat Mahjabeen

Genetic and expressional variations of APEX1 are associated with increased risk of head and neck cancer

The aetiology of head and neck cancer (HNC) has been shown to be associated with genetic and certain environmental factors that produce DNA damage. Base excision repair (BER) genes are responsible for repair of DNA damage caused by reactive oxygen species and other electrophiles and therefore are good candidate susceptibility genes for HNC. Apurinic/apyrimidinic endonuclease-1 (APEX1) proteins have important functions in the BER pathway. In this case-control study, all exons of the APEX1 gene and its exon/intron boundaries were amplified in 300 HNC cases and 300 matched healthy controls and then analysed by single-stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analysed. To confirm our observations, we examined APEX1 expression at mRNA level on 50 head and neck squamous cell carcinoma (HNSCC) and 50 normal control samples by quantitative real-time polymerase chain reaction. At germ line level, three novel mutations (13T > G, Ser129Arg and Val131Gly) of APEX1 were observed. The homozygous and heterozygous genotypes of APEX1 13T > G, Ser129Arg and Val131Gly appear to be significantly involved in the development of HNC. In the case of expressional level, APEX1 mRNA expression was positively correlated with tumour size, clinical stage and positive lymph node metastasis. Statistical analysis showed a significantly higher APEX1 mRNA level in HNC tumour tissue than in control samples. Our study demonstrated that APEX1 mutations and deregulation of APEX1 are associated with increased risk of HNC in the Pakistani population.

Mutational spectrum of Gelsolin and its down regulation is associated with breast cancer.

Cytoskeletal rearrangement occurs in variety of cellular processes and involves a wide spectrum of proteins. Gelsolin super family proteins control actin organization by severing and capping filament ends and nucleating actin assembly. Gelsolin is the founding member of this family and plays important role in pathogenesis of human neoplasia. This study aimed to investigate the germline mutations and expressional profile of Gelsolin in human breast cancer tissues. For germ line screening PCR-SSCP technique was used while expression was analyzed through quantitative real time PCR. Different types of mutations were observed in Gelsolin coding regions on exons 4, 10, 11, 14 and 15. These mutations include 3 missense nonsynonymous substitution mutations, 2 deletions, 1 insertion and 1 synonymous substitution mutation. Gelsolin transcript level was found significantly lower in breast tumor tissues compared to control samples (p=0.03). Low level of Gelsolin was found in metastatic patients (p=0.002) and patients who died from breast cancer (P=0.03) compared to disease free patients at final follow up. This study shows that level of Gelsolin is down regulated in breast cancer tissues and is linked with metastasis development and death in patients. It is concluded that genetic changes in coding regions of Gelsolin can potentially contribute to genetic instability. These genetic variations and expressional correlation with patient survival may prove to be of significant importance.

Correlation between selected XRCC2, XRCC3 and RAD51 gene polymorphisms and primary breast cancer in women in Pakistan.

Genetic polymorphisms in homologous recombination repair genes cause an abnormal development of cancerous cells. In the present study we evaluated the possibility of breast cancer association with single nucleotide polymorphisms of RAD51, XRCC2 and XRCC3 genes. Polymorphisms selected in this study were RAD51 135G/C, XRCC2 Arg188His; and XRCC3 Thr241Met. Each polymorphism was genotyped using Polymerase chain reaction-restriction fragment length polymorphism in study cohort of 306 females (156 breast cancer patients and 150 controls). We observed that heterozygous variant genotype (GC) of RAD51 135 G/C polymorphism was associated with a significantly (OR=2.70; 95%CI (0.63-1.79); p<0.03) increased risk of breast cancer. In case of the XRCC3 gene we observed that frequency of heterozygous (OR=2.88; 95%CI (1.02-8.14); p<0.02) and homozygous (OR=1.46; 95%CI (0.89-2.40); p<0.04) genotype of Thr241Met polymorphism were significantly higher in breast cancer patients. For the Arg188His polymorphism of XRCC2, ~2fold increase in breast cancer risk (OR=1.6, 95%CI = 0.73-3.50) was associated with GA genotype with a p value for trend of 0.03. Our results suggest that the 135G/C polymorphism of the RAD51, Thr241Met polymorphism of XRCC3 and Arg188His polymorphism of XRCC2 can be independent markers of breast cancer risk in Pakistan.

Association of Promoter Polymorphisms in Xrcc2 Gene Involved in DNA Double Strand Break Repair and Increased Susceptibility to Thyroid Cancer Risk in Pakistani Population

Introduction: The incidence of thyroid cancer (TC) has rapidly increased globally in recent decades. It is the most frequent endocrine malignancy which is fifth most common cancer in females. Double strand break repair (DSBR) pathway gene, X-Ray Repair Complementing Defective Repair in Chinese Hamster Cells 2 (XRCC2) has high rate of polymorphisms and may cause individual’s susceptibility towards carcinogenesis including thyroid cancer. Objective: Main objective of present study is to find the association of hotspot promotor polymorphisms in XRCC2 gene with thyroid cancer risk. Methods: In this study, we performed genetic association studies in 856 individuals (456 cases and 400 controls) for three promoter region SNPs of XRCC2 gene i.e., G4234C (rs3218384), G4088T (rs3218373) and G3063A (rs2040639). Genotyping was performed by amplification refractory mutation system (ARMS-PCR) followed by direct sequencing. Results: We found association of G4234C with thyroid cancer risk in stage I and II (p>0.0004) cancer patients while no association was observed with other parameters. Significant increased risk of developing thyroid cancer risk was observed in patients for G4088T with variant heterozygote T/G (OR=1.65, 95% CI=1.20-2.24; p<0.001) and polymorphic homozygote G/G (OR=1.66, 95% CI=1.16-2.36; p=0.005) compared with healthy controls. For G3063A polymorphism, a significant difference in genotypes distributions was observed for heterozygous variant G/A (OR=2.11; 95% CI=1.52-2.94; p<0.0001) and A/A variant genotype (OR=2.02; 95% CI=1.37-2.97; p<0.0003). When stratified for different parameters, significant increased risk was observed in female patients, patients with age ≥ 42 years, smoking and stage I and II patients for G4088T and G3063A in comparison to controls. Conclusion: Present study concluded that G4234C, G4088T and G3063A SNPs in XRCC2 gene may modify the risk of thyroid cancer development.

Association of RAD 51 135 G/C, 172 G/T and XRCC3 Thr241Met Gene Polymorphisms with Increased Risk of Head and Neck Cancer

Homologous recombination repair (HRR) plays an important role in protection against carcinogenic factors. Genes regulating the HRR mechanisms may impair their functions and consequently result in increased cancer susceptibility. RAD 51 and XRCC3 are key regulators of the HRR pathway and genetic variability in these may contribute to the appearance and progression of various cancers including head and neck cancer (HNC). The aim of the present study was to compare the distribution of genotypes of RAD51 (135G/C, 172 G/T) and XRCC3 (Thr241Met) polymorphisms between HNC patients and controls. Each polymorphism was genotyped using the polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP) technique in 200 pathologically confirmed HNC patients along with 150 blood samples from normal, disease free healthy individuals. We observed that homozygous variant CC genotype of RAD51 135G/C was associated with a 2.5 fold increased HNC risk (OR=2.5; 95%CI=0.69-9.53; p<0.02), while second polymorphism of RAD 51 172 G/T, heterozygous variant GT genotype was associated with a 1.68 fold (OR=1.68; 95%CI=1.08-2.61; p<0.02) elevation when compared with controls. In the case of the Thr241Met polymorphism of XRCC3, we observed a 16 fold (OR=16; 95% CI= 3.78-69.67; p<0.0002) increased HNC risk in patients compared to controls. These results further suggested that RAD51 (135G/C, 172 G/T) and XRCC3 (Thr241Met) polymorphisms may be effective biomarkers for genetic susceptibility to HNC. Larger studies are needed to confirm our findings and identify the underlying mechanisms.

Loss of Mitochondrial Tumor Suppressor Genes Expression Is Associated with Unfavorable Clinical Outcome in Head and Neck Squamous Cell Carcinoma: Data from Retrospective Study.

Mitochondrial genes play important roles in cellular energy metabolism, free radical generation, and apoptosis. Dysregulation of these genes have long been suspected to contribute to the generation of reactive oxygen species (ROS), increased proliferation and progression of cancer. A family of orthologues of yeast silent information regulator 3 (SIRT3), 4 (SIRT4) and mitochondrial tumor suppressor 1 (MTUS1) are important mitochondrial tumor suppressor genes which play an important role in the progression of multiple cancers. However, their role in the development of oxidative stress, enhanced proliferation and progression of head and neck squamous cell carcinoma (HNSCC) has not yet been studied. In this study we aimed to test the association between reduced mitochondrial tumor suppressor genes’ activities and enhancement in tissue oxidative stress and cell proliferation in HNSCC cases. The expression of mitochondrial tumor suppressor genes (SIRT3, SIRT4 and MTUS1), mitochondrial DNA repair gene (OGG1-2a) and a proliferation marker (Ki-67) was studied in a study cohort of 120 HNSCC patients and controls with reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR (qPCR) in order to determine the potential prognostic significance of these genes. A statistically significant downregulation of SIRT3 (p<0.001), SIRT4 (p<0.0001), MTUS1 (p<0.002) and OGG1 (p<0.0001) was observed in HNSCC compared to control samples. Ki-67 was also overexpressed (p<0.0001) in HNSCC versus control samples. Additionally, to explore gene–gene relationship, we observed a positive spearmen correlation between SIRT3 versus SIRT4 (r = 0.523***, p<0.0001), SIRT3 versus MTUS1 (r = 0.273***, p<0.001), SIRT3 versus OGG1-2a (r = 0.213*, p<0.03), SIRT4 versus OGG1 (r = 0.338***, p<0.0001) and MTUS1 versus OGG1-2a (r = 0.215*, p<0.03) in HNSCC cases. A negative spearman correlation was observed between OGG1 versus Ki-67 (r = -0.224**, p<0.01) and OGG1-2a versus Ki-67 (r = -0.224**, p<0.01) in HNSCC cases. Here we report that the deregulation of mitochondrial tumor suppressor genes (SIRT3, SIRT4 and MTUS1) in relation to decreased expression of mitochondrial DNA repair gene OGG1-2a and increased proliferation (measured by proliferation marker Ki-67) may be considered important factors in the development of head and neck squamous cell carcinoma.

Expression of CYP1A1 and GSTP1 in Human Brain Tumor Tissues in Pakistan

Most of the exogenous and endogenous chemical compounds are metabolized by enzymes of xenobiotic processing pathways, including the phase I cytochrome p450 species. Carcinogens and their metabolites are generally detoxified by phase II enzymes like glutathione-S-transferases (GST). The balance of enzymes determines whether metabolic activation of pro-carcinogens or inactivation of carcinogens occurs. Under certain conditions, deregulated expression of xenobiotic enzymes may also convert endogenous substrates to metabolites that can facilitate DNA adduct formation and ultimately lead to cancer development. In this study, we aimed to test the association between deregulation of metabolizing genes and brain tumorigenesis. The expression profile of metabolizing genes CYP1A1 and GSTP1 was therefore studied in a cohort of 36 brain tumor patients and controls using Western blotting. In a second part of the study we analyzed protein expression of GSTs in the same study cohort by ELISA. CYP1A1 expression was found to be significantly high (p<0.001) in brain tumor as compared to the normal tissues, with ~4 fold (OR=4, 95%CI=0.43-37) increase in some cases. In contrast, the expression of GSTP1 was found to be significantly low in brain tumor tissues as compared to the controls (p<0.02). This down regulation was significantly higher (OR=0.05, 95%CI=0.006-0.51; p<0.007) in certain grades of lesions. Furthermore, GSTs levels were significantly down-regulated (p<0.014) in brain tumor patients compared to controls. Statistically significant decrease in GST levels was observed in the more advanced lesions (III-IV, p<0.005) as compared to the early tissue grades (I-II). Thus, altered expression of these xenobiotic metabolizing genes may be involved in brain tumor development in Pakistani population. Investigation of expression of these genes may provide information not only for the prediction of individual cancer risk but also for the prevention of cancer.

Association of SYK genetic variations with breast cancer pathogenesis.

Spleen tyrosine kinase (SYK) is a non-receptor type cytoplasmic protein and a known tumor suppressor gene in breast cancer. Polymorphisms in SYK have been reported to be associated with cell invasion/cell morality and an increased risk of cancer development. In this case control study, all exons of the SYK gene and its exon/ intron boundaries were amplified in 200 breast cancer cases and 100 matched controls and then analyzed by single stranded conformational polymorphism. Amplified products showing altered mobility patterns were sequenced and analyzed. Twelve variations were identified in exonic and intronic regions of DNA encoding SH2 domain and kinase domain of the SYK gene. All of these mutations are novel. Among them, 5 missense mutations were observed in exon 15 while one missense mutation was found in exon 8. In addition to these mutations, six mutations were also identified in intronic regions. We found a significant association between SYK mutations and breast cancer and observed that Glu241Arg, a missense mutation is associated with an increase risk of ~7 fold (OR=6.7, 95% CI=1.54-28.8), Thr581Pro (missense mutation) is associated with increased risk of ~16 fold (OR=15.5, 95%CI=2.07-115.45) and 63367 T>G (missense mutation) is associated with increased risk of ~13 fold (OR=12.8, 95%CI=1.71-96.71) for breast cancer. Significant associations were observed for each of these variations with both late menopause (p<0.01) and early menarche (p<0.005) cases when compared to controls. Our findings suggest that the polymorphic gene SYK may contribute to the development of breast cancer in at least the Pakistani population. This study provides an insight view of SYK which may provide a significant finding for the pharmaceutical and biotechnology industry.

Novel germline CDK4 mutations in patients with head and neck cancer

BackgroundCyclin-dependent kinase 4 (CDK4) together with its regulatory subunit cyclin D1, governs cell cycle progression through G1 phase. Cyclin-dependent kinase inhibitors, including p16INK4A in turn regulate CDK4. In particular, deregulation of the p16/CDK4/cyclin D1 complex has been established in a variety of human tumors including gliomas, sarcomas, melanoma, breast and colorectal cancer. However, changes in CDK4 have rarely been observed.MethodIn this study we used a combination of PCR-SSCP and direct sequencing for mutational screening of CDK4. DNA was isolated from peripheral blood leukocyte of patients with squamous cell carcinoma of head and neck, for screening germline mutations in coding regions of CDK4.ResultsVariations observed in exon 2 and 5 were three missense mutations, g5051G > C (Ser52Thr), g5095G > C (Glu67Gln), g5906C > A, g5907C > G (Pro194Ser) and novel frame shift mutations g7321_23delTGA, g7121_7122insG, g7143delG in exon 7 and 3′UTR respectively.ConclusionIn conclusion, two novel mutations were found in N terminal domain which indicates that CDK4 mutation may play a major role in the development and progression of squamous cell carcinoma of head and neck.

OGG1 Mutations and Risk of Female Breast Cancer: Meta-Analysis and Experimental Data

In first part of this study association between OGG1 polymorphisms and breast cancer susceptibility was explored by meta-analysis. Second part of the study involved 925 subjects, used for mutational analysis of OGG1 gene using PCR-SSCP and sequencing. Fifteen mutations were observed, which included five intronic mutations, four splice site mutations, two 3′UTR mutations, three missense mutations, and a nonsense mutation. Significantly (p < 0.001) increased (~29 fold) breast cancer risk was associated with a splice site variant g.9800972T>G and 3′UTR variant g.9798848G>A. Among intronic mutations, highest (~15 fold) increase in breast cancer risk was associated with g.9793680G>A (p < 0.009). Similarly ~14-fold increased risk was associated with Val159Gly (p < 0.01), ~17-fold with Gly221Arg (p < 0.005), and ~18-fold with Ser326Cys (p < 0.004) in breast cancer patients compared with controls, whereas analysis of nonsense mutation showed that ~13-fold (p < 0.01) increased breast cancer risk was associated with Trp375STOP in patients compared to controls. In conclusion, a significant association was observed between OGG1 germ line mutations and breast cancer risk. These findings provide evidence that OGG1 may prove to be a good candidate of better diagnosis, treatment, and prevention of breast cancer.